Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that regulate key cellular processes including cell cycle progression and RNA transcription (Shapiro G I. J Clin Oncol. 2006 Apr. 10; 24(11):1770-83). Heterodimerized with regulatory cyclin units, CDKs can be generally divided into two groups based on their functions. The first group consists of core cell cycle components and governs the cell cycle transition and cell division: cyclin D-dependent kinases 4/6 and cyclin E-dependent kinase 2, which control the G1→S transition; cyclin A-dependent kinases 1/2, a critical regulator of S-phase progression; cyclin B-dependent CDK1, required for the G2→M transition; and cyclin H/CDK7, the CDK-activating kinase. The second group, so called transcriptional CDKs, includes cyclin H/CDK7 and cyclin T/CDK9 which phosphorylate the C-terminal domain (CTD) of RNA polymerase II and promote transcriptional initiation and elongation.
The deregulation of the CDK activity is detected in virtually all forms of human cancer, most frequently due to the overexpression of cyclins and loss of expression of CDK inhibitors (de Cárcer G et al., Curr Med Chem. 2007; 14(9):969-85). CDK4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo (Lukas J et al., Nature. 1995 Jun. 8; 375(6531):503-6; Schreiber M et al., Oncogene. 1999 Mar. 4; 18(9):1663-76; Fry D W et al., Mol Cancer Ther. 2004 November; 3(11):1427-38). Various approaches aimed at targeting CDK2/1 have been reported to induce S and G2 arrest followed by apoptosis (Chen Y N et al., Proc Natl Acad Sci USA. 1999 Apr. 13; 96(8):4325-9; Chen W et al., Cancer Res. 2004 Jun. 1; 64(11):3949-57; Mendoza N et al., Cancer Res. 2003 Mar. 1; 63(5):1020-4). Inhibition of the transcriptional CDKs 7 and 9 can affect the accumulation of transcripts encoding anti-apoptosis family members, cell cycle regulators, as well as p53 and NF-κB-responsive gene targets (Lam L T et al., Genome Biol. 2001; 2(10):RESEARCH0041). All these effects contribute to the induction of apoptosis and also potentiation of cytotoxicity mediated by disruption of a variety of pathways in many cancer cell types (Chen R et al., Blood. 2005 Oct. 1; 106(7):2513-9; Pepper C et al., Leuk Lymphoma. 2003 February; 44(2):337-42). CDKs are therefore recognized as an attractive target for the design and development of compounds that can specifically bind and inhibit the cyclin-dependent kinase activity and its signal transduction pathway in cancer cells, and thus can serve as either diagnostic or therapeutic agents. For example, the potent and highly selective CDK2/1 inhibitor, SNS-032 (BMS-387032), and the CDK4/6 inhibitor, PD 332991, are currently in clinical trials for treatment of cancer.
Numerous reports have indicated that CDK inhibitors may be therapeutically effective in several other disease indications than cancer, including polycystic kidney disease (Ibraghimov-Beskrovnaya O, Cell Cycle. 2007, 6:776-9), mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, proliferative lupus nephritis, collapsing glomerulopathy, IgA nephropathy (Soos T J et al., Drug News Perspect. 2006, 19:325-8) and Alzheimer's disease (Monaco E A & Vallano M L. Front Biosci. 2005. 10:143-59). CDKs are required for replication of many viruses such as human cytomegalovirus, herpes simplex virus type 1 and HIV-1. Specific pharmacological CDK inhibitors have demonstrated broad antiviral activities (Schang L M et al., Antivir Chem Chemother. 2006; 17(6):293-320; Pumfery A et al., Curr Pharm Des. 2006; 12(16):1949-61).
Despite the early success of certain kinase inhibitors, it has become clear that selectively targeting individual kinases can lead to the development of drug resistant tumors. Cells that have developed mutations within the drug/kinase binding pocket display a growth advantage in the presence of drug eventually leading to disease progression. Current clinical strategies aimed at combining these molecularly targeted drugs with standard chemotherapeutics, radiation, or other targeted agents will lead to novel strategies to improve overall response rate and increase the number of complete remissions.
Furthermore, elucidation of the complex and multifactorial nature of various diseases that involve multiple pathogenic pathways and numerous molecular components suggests that multi-targeted therapies may be advantageous over mono-therapies. Recent combination therapies with two or more agents for many such diseases in the areas of oncology, infectious disease, cardiovascular disease and other complex pathologies demonstrate that this combinatorial approach may provide advantages with respect to overcoming drug resistance, reduced toxicity and, in some circumstances, a synergistic therapeutic effect compared to the individual components. Certain cancers have been effectively treated with such a combinatorial approach; however, treatment regimes using a cocktail of cytotoxic drugs often are limited by dose limiting toxicities and drug-drug interactions. More recent advances with molecularly targeted drugs have provided new approaches to combination treatment for cancer, allowing multiple targeted agents to be used simultaneously, or combining these new therapies with standard chemotherapeutics or radiation to improve outcome without reaching dose limiting toxicities. However, the ability to use such combinations currently is limited to drugs that show compatible pharmacologic and pharmacodynamic properties. In addition, the regulatory requirements to demonstrate safety and efficacy of combination therapies can be more costly and lengthy than corresponding single agent trials. Once approved, combination strategies may also be associated with increased costs to patients, as well as decreased patient compliance owing to the more intricate dosing paradigms required.
In the field of protein and polypeptide-based therapeutics it has become commonplace to prepare conjugates or fusion proteins that contain most or all of the amino acid sequences of two different proteins/polypeptides and that retain the individual binding activities of the separate proteins/polypeptides. This approach is made possible by independent folding of the component protein domains and the large size of the conjugates that permits the components to bind their cellular targets in an essentially independent manner. Such an approach is not, however, generally feasible in the case of small molecule therapeutics, where even minor structural modifications can lead to major changes in target binding and/or the pharmacokinetic/pharmacodynamic properties of the resulting molecule.
The use of CDK inhibitors in combination with histone deacetylases (HDAC) has been shown to produce synergistic effects. Histone acetylation is a reversible modification, with deacetylation being catalyzed by a family of enzymes termed HDAC's. HDAC's are represented by X genes in humans and are divided into four distinct classes (J Mol Biol, 2004, 338:1, 17-31). In mammalians class I HDAC's (HDAC1-3, and HDAC8) are related to yeast RPD3 HDAC, class 2 (HDAC4-7, HDAC9 and HDAC10) related to yeast HDA1, class 4 (HDAC11), and class 3 (a distinct class encompassing the sirtuins which are related to yeast Sir2).
Histones are subject to post-translational acetylation of the, ε-amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1) (Csordas, Biochem. J., 1990, 286: 23-38). Acetylation neutralizes the positive charge of the lysine side chain, and is thought to impact chromatin structure. Indeed, access of transcription factors to chromatin templates is enhanced by histone hyperacetylation, and enrichment in underacetylated histone H4 has been found in transcriptionally silent regions of the genome (Taunton et al., Science, 1996, 272:408-411). In the case of tumor suppressor genes, transcriptional silencing due to histone modification can lead to oncogenic transformation and cancer.
Several classes of HDAC inhibitors currently are being evaluated by clinical investigators. The first FDA approved HDAC inhibitor is Suberoylanilide hydroxamic acid (SAHA, Zolinza®) for the treatment of cutaneous T-cell lymphoma (CTCL). Other HDAC inhibitors include hydroxamic acid derivatives; PXD101 and LAQ824, are currently in the clinical development. In the benzamide class of HDAC inhibitors, MS-275, MGCD0103 and CI-994 have reached clinical trials. Moume et al. (Abstract #4725, AACR 2005), demonstrate that thiophenyl modification of benzamides significantly enhance HDAC inhibitory activity against HDAC1.
Recent advances suggest that CDK inhibitors in combination with HDAC inhibitors may provide advantageous results in the treatment of cancer. For example, HDAC inhibitor Valproic acid upregulated p16INK4A, a CDK inhibitor, and induced apoptosis in melanoma cell lines (Valentini A et al., Cancer Biol Ther. 2007 February; 6(2):185-91). Trichostatin A induced cyclin D1 repression contributed to the inhibition of breast cancer cell proliferation and sensitized cells to CDK inhibitors (Alao J P et al., Mol Cancer. 2006 Feb. 20; 5:8). Co-administration of flavopiridol, a pan-CDK inhibitor, with HDAC inhibitors synergistically potentiated mitochondrial damage, caspase activation and cell death in human leukemia cells (Dasmahapatra G et al., Mol Pharmacol. 2006 January; 69(1):288-98). Combination treatment of HDAC and CDK inhibitors has now entered clinical arena in patients with leukemia and other hematologic malignancies (Grant S and Dent P, Curr. Drug Targets. 2007 June; 8(6):751-9).
Current therapeutic regimens of the types described above attempt to address the problem of drug resistance by the administration of multiple agents. However, the combined toxicity of multiple agents due to off-target side effects as well as drug-drug interactions often limits the effectiveness of this approach. Moreover, it often is difficult to combine compounds having differing pharmacokinetics into a single dosage form, and the consequent requirement of taking multiple medications at different time intervals leads to problems with patient compliance that can undermine the efficacy of the drug combinations. In addition, the health care costs of combination therapies may be greater than the cost of single molecule therapies. Furthermore, it may be more difficult to obtain regulatory approval of a combination therapy since the burden for demonstrating activity/safety of a combination of two agents may be greater than for a single agent (Dancey J & Chen H, Nat. Rev. Drug Dis., 2006, 5:649). The development of novel agents that target multiple therapeutic targets selected not by virtue of cross reactivity, but through rational design will help improve patient outcome while avoiding these limitations. Thus, enormous efforts are still directed to the development of selective anti-cancer drugs as well as to new and more efficacious combinations of known anti-cancer drugs.